The primary aim of this proposal is to study the nature of the early binding process of SP82G phage DNA in transfection and DNA marker rescue in B. subtilis. The early extracellular binding of DNA is a complex sequence which involves specific binding of each end of the infecting molecule to the competent bacterium in two separate events prior to the attainment of DNAse resistance. Entry into a DNAse resistant form occurs in a uniquely polar order. Molecular binding specificity occurs in each of these events and it is our intention to biochemically dissect the competent B. subtilis cell to determine what cellular components and types of bonds are involved in the binding of SP82G DNA. Radioisotope labeling techniques and genetic analysis will be used to identify both bulk and site-specific DNA associated with the early complex components. As well, bacterial mutants which are defective in transformation will be analyzed to determine in which, if any, of the early extracellular binding events they are defective. Among the steps which might be defective in the transformation mutants are: the ability to form the initial reversible binding, the ability to convert this binding to a heat irreversible bond involving the right end of the molecular, the ability to bind the left end of the molecule to a cold sensitive element, and lastly the ability to transport the DNA to a DNAse resistant state. Further we propose to continue our efforts to define the relationship of these binding events to the overall DNA infection process and to study, in particular, the bacterial intracellular inactivation mechanism, and the relationship between this cellular property and competence. We wish as well to continue studies on the relationship between the SP82G DNA molecule and its genetic map.